Recombinant Neosartorya fischeri Probable carboxypeptidase NFIA_052450 (NFIA_052450)

What is Recombinant Neosartorya fischeri Probable carboxypeptidase NFIA_052450 and what is its significance in research?

Recombinant Neosartorya fischeri Probable carboxypeptidase NFIA_052450 is a protein from the fungus Neosartorya fischeri (also known as Aspergillus fischeri) produced through recombinant DNA technology, typically in E. coli expression systems . It belongs to the carboxypeptidase family of enzymes, which perform diverse physiological functions by removing C-terminal amino acids from proteins and peptides .

The significance of this protein in research stems from several areas:

• As a member of the metallocarboxypeptidase family, it may have roles in protein processing and modification

• Potential applications in understanding fungal physiology and metabolism

• Possible relevance to the development of antifungal strategies, given that other proteins from N. fischeri have demonstrated antifungal properties

Unlike the well-characterized antifungal proteins NFAP and NFAP2 from the same organism, NFIA_052450 remains less studied but represents an important target for enzyme research and potential biotechnological applications.

What are the general functions of carboxypeptidases in fungi like Neosartorya fischeri?

Carboxypeptidases in fungi like Neosartorya fischeri perform several critical physiological functions:

• Protein Processing: They participate in post-translational modification by removing C-terminal amino acids from proteins and peptides .

• Nutrient Acquisition: In fungi, these enzymes can facilitate the breakdown of extracellular proteins into assimilable nitrogen sources.

• Cellular Metabolism: They may be involved in the regulation of protein turnover and homeostasis.

• Specialized Functions: Some fungal carboxypeptidases have evolved specialized roles, such as participating in the production of bioactive compounds .

The diversity of carboxypeptidases in fungi suggests their adaptation to various cellular processes and environmental conditions. In N. fischeri specifically, other enzymes such as β-glucosidase (NfBGL1) have been characterized with roles in substrate hydrolysis , indicating a diverse enzymatic repertoire in this organism that likely includes specialized carboxypeptidases.

How can researchers express and purify Recombinant NFIA_052450 for functional studies?

Expression and purification of Recombinant NFIA_052450 can be approached using methodologies demonstrated effective for other Neosartorya fischeri proteins:

Expression Systems:

• E. coli: Most commonly used for NFIA_052450 expression

• Pichia pastoris: Successfully used for other N. fischeri proteins like NFAP

• Penicillium chrysogenum-based system: Shown to be effective for NFAP2 expression

Recommended Expression Protocol:

• Clone the NFIA_052450 gene into an appropriate expression vector

• Transform the vector into the chosen expression host

• Induce protein expression (e.g., with IPTG for E. coli or methanol for P. pastoris)

• Harvest cells and extract the recombinant protein

Purification Strategy:

• Initial clarification of cell lysate

• Affinity chromatography (if the protein is tagged)

• Ion-exchange chromatography for further purification

• Size-exclusion chromatography as a polishing step

• Heat treatment may be applicable if the protein is thermostable, as observed with other N. fischeri proteins

Verification Methods:

• SDS-PAGE to confirm protein size

• Western blotting for identity confirmation

• Mass spectrometry for accurate molecular weight determination

• Activity assays to confirm enzymatic function

For optimal results, consider including a purification tag (His-tag, GST, etc.) in the recombinant construct to facilitate affinity purification, with an option for tag removal if needed for functional studies.

What methods are suitable for characterizing the enzymatic activity of recombinant NFIA_052450?

Characterizing the enzymatic activity of recombinant NFIA_052450 requires targeted approaches based on its predicted carboxypeptidase function:

Substrate Selection:

• Synthetic peptides with C-terminal modifications

• Fluorogenic or chromogenic substrates for continuous monitoring

• Natural protein substrates to assess physiological relevance

Activity Assay Methods:

• Spectrophotometric assays: Monitor the release of amino acids with specific reagents

• Fluorometric assays: Using fluorescence-quenched substrates

• HPLC-based assays: For precise quantification of cleaved products

• Mass spectrometry: For detailed substrate specificity analysis

Optimal Assay Conditions Determination:

• pH optimization (typically test range pH 4.0-9.0)

• Temperature profiling (25-80°C based on N. fischeri enzyme thermostability)

• Metal ion requirements (test Zn²⁺, Ca²⁺, Mg²⁺, Mn²⁺)

• Buffer composition effects

Kinetic Parameter Determination:
Employ Multiple Injection Method (MIM) with Isothermal Titration Calorimetry (ITC) as described for enzyme kinetics :

For optimal experimental design, implement the penalized expectation of determinant (ED)-optimal design approach to reduce the uncertainty of parameter estimates , which has been shown to improve the Cramer-Rao lower bound of the variance of parameter estimation error to 82% for μmax and to 60% for KM compared to batch experiments.

How does NFIA_052450 compare structurally and functionally to other characterized proteins from Neosartorya fischeri?

While specific structural and functional data for NFIA_052450 is limited, we can draw comparisons with other characterized proteins from Neosartorya fischeri:

Structural Comparison:

Functional Comparison:

• NFIA_052450: Probable carboxypeptidase activity, specific substrates unknown

• NFAP: Antifungal activity against filamentous fungi, disrupts cell wall organization

• NFAP2: Potent anti-Candida activity, potential clinical applications

• NfBGL1: β-glucosidase activity, converts isoflavone glycosides to aglycones

Evolutionary Context:
The diverse functional profiles of proteins from N. fischeri suggest adaptation to various ecological niches. While NFAP and NFAP2 appear specialized for antimicrobial defense, NFIA_052450 likely serves a different physiological role, potentially in metabolism or protein processing.

Unlike the well-characterized antifungal proteins, NFIA_052450's specific function remains to be elucidated through detailed biochemical characterization, but methods deployed for other N. fischeri proteins provide a roadmap for its investigation.

What challenges might researchers face during heterologous expression of NFIA_052450?

Researchers expressing NFIA_052450 heterologously may encounter several challenges based on experiences with other Neosartorya fischeri proteins:

Expression Yield Limitations:

• Low-yield production has been reported as a limiting factor for native N. fischeri proteins

• The average yield of recombinant NFAP2 was 40-times higher than in the native producer, indicating optimization is essential

Protein Folding and Processing:

• Correct disulfide bond formation may be critical if NFIA_052450 contains multiple cysteine residues (as seen with NFAP)

• Post-translational modifications present in the native protein may be absent in prokaryotic expression systems

Challenges by Expression System:

Purification Challenges:

• Proteolytic degradation during extraction

• Co-purification of host cell proteins with similar properties

• Maintaining enzyme activity during purification steps

Solutions from Literature:
Studies with NFAP and NFAP2 have overcome these challenges through:

• Codon optimization for expression hosts

• Use of fusion tags to enhance solubility and facilitate purification

• High-cell-density fermentation techniques yielding up to 1873±1.5 U/ml activity

• Development of alternative production methods such as synthetic peptide synthesis and native chemical ligation

A systematic approach to expression optimization is recommended, testing multiple expression constructs, hosts, and conditions in parallel.

How can researchers design optimal experiments to determine the kinetic parameters of NFIA_052450?

Determining kinetic parameters of NFIA_052450 requires sophisticated experimental design to ensure accurate and reliable results:

Optimal Experimental Design Strategy:

• Preliminary Experimental Planning:

  • Employ penalized expectation of determinant (ED)-optimal design with discrete parameter distribution

  • Optimize sample times and starting concentrations (C₀) to minimize uncertainty of parameter estimates

  • For substrate feed-batch process design, implement small volume flow which has been shown to reduce parameter estimation error compared to batch experiments

• Calorimetric Approach:

  • Utilize Isothermal Titration Calorimetry (ITC) with Multiple Injection Method (MIM)

  • Design two experiments:

    • Experiment 1: Determine dQ/dt by titrating substrate into limited enzyme

    • Experiment 2: Determine reaction enthalpy by converting all substrate to product

• Data Collection Parameters:

• Data Analysis Framework:

  • Plot reaction rate (d[P]/dt) versus substrate concentration [S]

  • Fit to Michaelis-Menten equation: v = (Vmax × [S])/(KM + [S])

  • Analyze with Lineweaver-Burk double reciprocal plot for visualization of inhibition patterns

  • Calculate kcat from Vmax/[E]total

• Validation Approaches:

  • Perform experiments at multiple enzyme concentrations

  • Compare results from different methodologies (spectrophotometric, fluorometric)

  • Assess impact of potential inhibitors and activators

This approach has been shown to reduce the Cramer-Rao lower bound of the variance of parameter estimation error to 82% for μmax and to 60% for KM compared to conventional batch experiments .

What techniques are most appropriate for investigating the substrate specificity of NFIA_052450?

Investigating the substrate specificity of NFIA_052450 requires a multi-faceted approach to understand both the range of acceptable substrates and the structural determinants of specificity:

Comprehensive Substrate Profiling:

• Peptide Library Screening:

  • Utilize positional scanning synthetic combinatorial libraries

  • Test peptides with varying C-terminal residues to determine P1' preferences

  • Analyze upstream residue preferences (P1, P2, P3) using systematically varied peptides

• Carboxypeptidase B-Assisted Charge-Based Fractional Diagonal Chromatography (CPB-ChaFRADIC):

  • Adapt methodology from result for specificity analysis

  • This technique reveals cleavage preferences through selective modification of C-terminal amino acids

  • Has demonstrated high efficiency and no deficiency for His/Lys/Arg-containing C-termini

• Mass Spectrometry-Based Approaches:

  • Perform LC-MS/MS analysis of digestion products

  • Identify cleavage sites through comparative peptide mapping

  • Quantify relative cleavage efficiencies with isotope-labeled substrates

Structural Basis of Specificity:

• Homology Modeling and Docking Studies:

  • Generate structural models based on related carboxypeptidases

  • Identify putative substrate binding pockets and catalytic residues

  • Perform in silico docking to predict substrate interactions

• Site-Directed Mutagenesis:

  • Mutate predicted substrate-binding residues

  • Analyze shifts in specificity or activity following mutations

  • Create a structure-function map of the substrate binding pocket

Experimental Validation Framework:

Based on observations with carboxypeptidase B, which selectively catalyzes the release of C-terminal lysine and arginine , particular attention should be paid to basic amino acids in the substrate screening, while recognizing that NFIA_052450 may have different specificity.

For comprehensive analysis, include examination of:

• pH dependence of substrate preferences

• Metal ion effects on specificity

• Temperature influence on substrate selection

• Competitive substrate assays to determine relative preferences

What role might NFIA_052450 play in the biology of Neosartorya fischeri, and how can this be investigated?

Understanding the biological role of NFIA_052450 in Neosartorya fischeri requires integrative approaches spanning genetics, biochemistry, and systems biology:

Hypothesized Biological Functions:

Based on carboxypeptidase roles in other systems and the biology of N. fischeri, NFIA_052450 might function in:

• Nutrient acquisition through protein degradation

• Cell wall remodeling during growth and development

• Post-translational processing of secreted proteins

• Detoxification of harmful peptides in the environment

• Regulation of signaling peptides

Investigative Approaches:

• Genetic Manipulation Studies:

  • Gene knockout/knockdown using CRISPR-Cas9 or RNAi

  • Overexpression analysis to observe gain-of-function phenotypes

  • Promoter-reporter fusions to determine expression patterns

  • Heterologous expression in a model organism like Aspergillus nidulans

• Biochemical and Proteomic Approaches:

  • Identify natural substrates through pull-down experiments

  • Characterize the secretome and peptidome in wild-type vs. NFIA_052450 mutants

  • Analyze changes in cell wall composition in mutants

  • Perform metabolomic profiling to identify pathways affected by NFIA_052450 activity

• Localization and Expression Studies:

  • Fluorescent protein tagging to determine subcellular localization

  • Immunolocalization with specific antibodies

  • qRT-PCR to measure expression under various conditions

  • RNA-seq to identify co-regulated genes

• Environmental Response Characterization:

• Comparative Genomics:

  • Analyze conservation of NFIA_052450 across fungal species

  • Identify syntenic relationships and gene neighborhoods

  • Examine evolutionary patterns suggestive of functional constraints

  • Compare with carboxypeptidases of known function in other organisms

For N. fischeri specifically, the thermal stability of its enzymes (with temperature optima up to 80°C for some enzymes ) suggests investigating how NFIA_052450 might contribute to thermoadaptation and survival in high-temperature environments.

How can site-directed mutagenesis be applied to investigate structure-function relationships in NFIA_052450?

Site-directed mutagenesis offers a powerful approach to dissect the structure-function relationships in NFIA_052450, providing insights into catalytic mechanism, substrate specificity, and protein stability:

Strategic Mutagenesis Targets:

• Catalytic Residues:

  • Identify putative catalytic triad/dyad based on homology to characterized carboxypeptidases

  • Create conservative mutations (e.g., Glu→Asp, His→Asn) to assess catalytic requirements

  • Generate complete knockout mutations to confirm essentiality

• Metal-Binding Sites:

  • If NFIA_052450 is a metallocarboxypeptidase, mutate predicted metal-coordinating residues

  • Analyze effects on activity, stability, and metal preference

  • Create variants with altered metal specificity

• Substrate-Binding Pocket:

  • Target residues lining the predicted S1' pocket that interacts with the C-terminal residue

  • Introduce mutations that alter pocket size, hydrophobicity, and charge

  • Engineer specificity shifts by rational design of binding site residues

• Structural Elements:

  • Following the approach used for NFAP , investigate:

    • Disulfide bridges through cysteine deletion mutants (NFAPΔC equivalent)

    • Hydrophobic core through hydrophobic residue deletion (NFAPΔh equivalent)

    • N-terminal domain through amino acid exchanges (NFAPΔN equivalent)

Mutagenesis Design Framework:

Integrated Analysis Approach:

• Functional Characterization:

  • Compare enzyme kinetics (kcat, KM) between wild-type and mutants

  • Assess substrate specificity changes using peptide libraries

  • Determine pH-activity and temperature-activity profiles

• Structural Verification:

  • Use circular dichroism or nuclear magnetic resonance to confirm structural integrity

  • Employ thermal shift assays to assess stability changes

  • Perform mass spectrometry to verify correct formation of disulfide bonds

• Computational Support:

  • Molecular dynamics simulations to predict effects of mutations

  • Homology modeling to visualize structural changes

  • Docking studies to predict altered substrate interactions

This comprehensive mutagenesis approach, similar to that applied to NFAP , will reveal which structural elements are critical for NFIA_052450's folding, stability, and catalytic function, providing a foundation for potential protein engineering applications.

How can researchers investigate potential applications of NFIA_052450 in biotechnology and medicine?

Investigating applications of NFIA_052450 in biotechnology and medicine requires systematic exploration of its properties and potential uses based on its enzymatic activity:

Biotechnological Applications Assessment:

• Biocatalysis Applications:

  • Screen activity on industrially relevant substrates

  • Evaluate performance under process-relevant conditions (temperature, pH, solvents)

  • Assess stability during prolonged reactions and storage

  • Compare efficiency to commercially used enzymes

• Food and Beverage Processing:

  • Test protein modification capabilities in food matrices

  • Evaluate flavor enhancement potential through peptide modification

  • Assess allergen reduction capabilities through specific peptide cleavage

  • Investigate texture modification properties

• Agricultural Applications:

  • Following studies with NFAP , evaluate:

    • Crop protection potential against fungal pathogens

    • Safety profile in plant models (e.g., Medicago truncatula)

    • Stability in field-relevant conditions

    • Compatibility with other agricultural treatments

Medical Applications Exploration:

• Therapeutic Potential Assessment:

  • Investigate selective proteolytic capabilities for:

    • Bioactive peptide production

    • Protein drug modification

    • Diagnostic applications

  • Screen for inhibition of pathologically relevant peptides

• Anti-Fungal Applications:

  • Based on NFAP2's anti-Candida activity :

    • Test NFIA_052450 against clinically relevant fungi

    • Evaluate synergy with conventional antifungals

    • Assess resistance development potential

    • Determine toxicity profile in human cell models

Safety and Production Considerations:

• Engineering for Application:

  • Develop immobilization strategies for reuse and stability enhancement

  • Engineer variants with improved properties (thermostability, pH tolerance)

  • Design fusion proteins for targeted applications

  • Optimize formulation for specific delivery methods

Drawing from the experience with NFAP and NFAP2, which demonstrated both agricultural and clinical potential , a parallel exploration of NFIA_052450's capabilities could reveal unique applications based on its carboxypeptidase activity.

What advanced techniques can be used to determine the three-dimensional structure of NFIA_052450?

Determining the three-dimensional structure of NFIA_052450 requires sophisticated structural biology techniques. Based on approaches used for other Neosartorya fischeri proteins, the following methods are recommended:

X-ray Crystallography Approach:

• Crystallization Screening:

  • Implement sparse matrix screening with commercial kits

  • Explore crystallization with bound substrates or inhibitors

  • Test seeding techniques to improve crystal quality

  • Consider surface entropy reduction mutations to enhance crystallizability

• Data Collection and Processing:

  • Collect high-resolution diffraction data at synchrotron facilities

  • Process data with modern software packages (XDS, DIALS)

  • Consider experimental phasing using heavy atoms if molecular replacement fails

  • Implement anisotropic diffraction correction if necessary

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Sample Preparation:

  • Produce isotopically labeled protein (¹⁵N, ¹³C, ²H)

  • Optimize buffer conditions for stability and spectral quality

  • Determine optimal temperature for data collection

• Spectral Assignment and Structure Calculation:

  • Collect suite of 2D and 3D heteronuclear experiments

  • Assign backbone and side-chain resonances

  • Measure distance restraints using NOE experiments

  • Calculate structural ensemble using restrained molecular dynamics

Complementary Techniques:

Integration of Structural and Functional Data:

From studies with NFAP, we know that disulfide bridges, hydrophobic core, and N-terminal amino acids play crucial roles in forming stable, folded, and functionally active proteins from N. fischeri . Similar structural elements should be carefully characterized in NFIA_052450 to understand their contribution to carboxypeptidase activity.

How does the stability and activity of NFIA_052450 compare across different environmental conditions?

Understanding the stability and activity profile of NFIA_052450 across various environmental conditions is crucial for both basic research and applications. Based on studies of other Neosartorya fischeri enzymes, a comprehensive characterization would include:

Temperature Effects:

• Thermal Stability Analysis:

  • Determine melting temperature (Tm) using differential scanning calorimetry

  • Assess activity retention after heat treatment at various temperatures

  • Measure half-life at elevated temperatures

  • Compare with other N. fischeri enzymes, which have shown remarkable thermostability (e.g., NfBGL1 with 80°C temperature optimum)

• Temperature-Activity Relationship:

  • Determine temperature optimum for enzymatic activity

  • Construct Arrhenius plot to calculate activation energy

  • Identify cold-activity properties if present

pH and Ionic Effects:

• pH Stability Profile:

  • Measure residual activity after incubation at various pH values

  • Determine pH stability range for storage and application

  • Compare with NFAP, which showed stability over a broad pH range (3.0-10.0)

• pH-Activity Relationship:

  • Determine optimal pH for enzymatic activity

  • Analyze pH-dependent changes in kinetic parameters

  • Identify ionizable groups involved in catalysis through pH-rate profiles

• Ionic Strength Effects:

  • Assess activity in presence of varying concentrations of salts

  • Test specifically the effect of mono- and divalent cations (K⁺, Na⁺, Mg²⁺)

  • Determine if, like NFAP, activity is affected by cations such as KCl, Mg(2)SO(4), Na(2)SO(4)

Solvent and Chemical Stability:

• Organic Solvent Tolerance:

  • Test activity retention in water-miscible organic solvents

  • Determine concentration thresholds for activity loss

  • Evaluate potential for biocatalysis in non-aqueous media

• Chemical Denaturant Resistance:

  • Measure activity in presence of urea or guanidinium chloride

  • Determine concentration causing 50% activity loss (C50)

  • Compare with NFAP's high resistance to denaturing conditions

Environmental Stability Matrix: